Probiotic bacteria are microorganisms that benefit the host by preventing or ameliorating disease. However, little information is known regarding the scientific rationale for using probiotics as alternative medicine. We reported that one such probiotic, Lactobacillus rhamnosus GG (LGG), prevents cytokine-induced apoptosis in both mouse and human colon cells through activating the anti-apoptotic Akt/protein kinase B and inhibiting cytokine-stimulated pro-apoptotic p38/mitogen-activated protein (MAP) activation. We further demonstrated that products recovered from LGG culture broth supernatant (LGG-s) show concentration-dependent activation of Akt and inhibition of cytokine-induced apoptosis. The long-term goal of this study is to investigate the mechanism of the probiotic bacterial regulatory effects on normal intestinal growth and development and to provide the mechanistic basis for potential therapeutic applications in diseases. The present research proposal is focused on studying the cellular and molecular mechanisms of probiotic regulation of intestinal epithelial cells homeostasis. Three Specific Aims are addressed: Aim 1. To identify proteins recovered from LGG-s that regulate colon epithelial cell survival. Proteins in LGG-s will be purified by column chromatography or electro-elution, and used to treat mouse and human colon epithelial cells to determine their effects on Akt activation and cell survival Aim 2. To determine amino acid sequence and clone the gene(s) encoding cell signaling and survival regulating proteins present in LGG-s. Amino acid sequences of purified proteins from LGG-s which regulate colon cell survival (Aim 1) will be determined for designing degenerate oligonucleotide probes. These probes will be used to screen/hybridize the LGG genomic DNA library to obtain DNA encoding the message for the full-length protein. Aim 3. To determine intestinal epithelial cell interacting proteins and molecular targets of purified proteins from LGG-s which regulate cell survival, Pull-down assays or protein arrays using proteins in LGG-s expressed as GST-fusion proteins will be performed to screen colon epithelial cellular lysates. Genes regulated by LGG in colon cells will be identified using DNA microarray. These findings will have significant relevance to further understanding the probiotic regulation of intestinal health.